The presence of diadenosine hexaphosphate (Ap6A) in chromaffin cells is described. The characterization of Ap6A has been accomplished by HPLC techniques, using three different elution conditions, rechromatography, and coelution with standards. Treatment with phosphodiesterase from Crotalus durissus produced AMP and adenosine pentaphosphate. The HPLC techniques described allowed the quantification of Ap6A in the picomole range. Chromaffin granules store Ap6A in a quantity of 48.5 +/- 9.7 nmol/mg protein, with a molar ratio ATP/Ap6A of 27. In chromaffin cells the Ap6A value was 1.46 +/- 0.32 nmol/10(6) cells. Diadenosine hexaphosphate was released from chromaffin cells by the action of carbachol and a value of 64 +/- 15 pmol/10(6) cells was obtained, which represents 4-5% of the total cellular content.